Interleukin 1 (IL-1) is a proinflammatory cytokine that is critical for the development and progression of multiple sclerosis (MS) in humans and experimental autoimmune encephalomyelitis (EAE) in mice. Although IL-1 induces expression of many cytokines via the activation of nuclear factor ?B, induction of several chemokines important for MS and EAE, such as RANTES and IP-10, requires the activation of an additional transcription factor, interferon regulatory factor-1 (IRF-1). Significantly, IRF-1 is activated in the brains of MS patients, IRF-1 knockout mice are protected from EAE, and EAE is regulated by IRF-1 expressed in the central nervous system (CNS). We have shown that IL-1 stimulates and upregulates sphingosine kinase 1 (SphK1), one of the two isoenzymes that generate sphingosine-1-phosphate (S1P), a lipid mediator that regulates diverse cellular processes important for inflammation. S1P is increased in the CSF of MS patients and induces astrogliosis. FTY720, a pro-drug that is phosphorylated by SphK2 to an S1P mimetic (FTY720-P) and after secretion targets four of the five S1P receptors, except S1P2, is currently in phase III clinical trials, and effectively reduces the frequency and severity of MS. FTY720 reduces MS symptoms, in part, by downregulating S1P1 on lymphocytes thus preventing their S1P-dependent egress from the lymph nodes. However, FTY720 also accumulates in the brain where it reduces astrogliosis; nevertheless, its effects in the CNS are incompletely understood. We made two intriguing observations: 1) S1P directly binds to cellular inhibitor of apoptosis 2 (cIAP2) inside cells and stimulates cIAP2-mediated K63 regulatory ubiquitination of IRF-1 and its activation. 2) In contrast to intracellular S1P, extracellular S1P suppresses nuclear translocation of IRF-1 and induction of RANTES and IP-10 expression via S1P2 in astrocytes. Thus, we hypothesize that S1P modulates brain inflammation by novel intracellular and extracellular mechanisms that regulate IRF-1 functions. This hypothesis has significant implications for understanding mechanisms of S1P, FTY720, and IRFs actions in astrocyte-mediated neuroinflammation and MS. We propose the following specific aims: Aim 1. Analyze the role of intracellular S1P as a cofactor for cIAP2-mediated ubiquitination of IRF-1. Aim 2. Identify the mechanisms by which extracellular S1P affects IRF-1 activation. Aim 3. Define the roles of S1P regulation of IRF-1 in vivo. This proposal will enhance understanding of how the intricate interplay between IL-1, S1P and its receptors in astrocytes regulates inflammation and could pave the way for the design of even more effective MS therapies. PUBLIC HEALTH RELEVANCE: FTY720-P, a sphingosine-1-phosphate (S1P) mimetic, effectively reduces symptoms of multiple sclerosis (MS). We found that S1P controls interferon regulatory factor-1 (IRF-1) activation by two novel opposing mechanisms. Since IRF-1 is activated in the brains of MS patients and IRF-1 knockout mice are protected from experimental autoimmune encephalomyelitis, it is important to understand mechanisms of by which S1P, FTY720-P, and IRF-1 regulate astrocyte-mediated neuroinflammation and MS.